The hope of success of therapeutic interventions in cancer largely relies on the possibility to distinguish, with high accuracy, even closely-related tumor types. Indeed, the identification of tumor specific signatures has been a major challenge of the last ten years to predict the responsiveness to a given therapeutic plan and to reduce the impact of side effects to be expected if unresponsive oncologic patients are being treated.
The SELEX technique refers to Systematic Evolution of Ligands by EXponential enrichment. Single-stranded oligonucleotides have the diversity characteristic both in molecular structure and function, thus, a random library of single oligonucleotides is synthesized for binding to a target protein on the membrane. The oligonucleotides bound non-specifically are washed away and the oligonucleotides bound specifically were eluted in denatured condition and collected. The oligonucleotides are amplified by PCR for further selection. The high affinity oligonucleotides, namely aptamers that have high affinity with the target proteins, can be selected from the initial library through amplification and selection over many cycles. In 1990, Tuerk and Gold selected Aptamers of T4 RNA polymerase by SELEX (Tuerk C and Gold L. 1990). Subsequently, Ellington and Szostak showed great interests in the application of aptamers in scientific research and production. Aptamers soon become a valuable research tool and show great application prospected in the fundamental research, drug selection and clinical diagnosis and therapy (Ellington and Szostak, 1990). At present, many kinds of aptamers have come into clinical test phase. For example, drugs for curing thrombus and inhibiting endometrium hyperplasic and angiogenesis (Green L S et al., 1995, Tasset D M, et al., 1997, Ruckman J et al., 1998).
An innovative aspect of the aptamers is their use in “target identification/validation” to identify various cell surface targets of a specific cellular state.
The U.S. Pat. No. 5,580,737 discloses a method for identifying nucleic acid ligands to a target molecule comprising contacting a mixture of nucleic acid with the target molecule, allowing the partitioning of increased affinity nucleic acid and then, contacting the increased affinity nucleic acid with non-target molecule. In particular ligand to theophylline and caffeine are described.
The patent application WO 2007/142713 provides a method for obtaining a probe specific for extracellular or cell-surface markers comprising several cycles of positive selection steps on a target cell followed by a step of counter-selection on a control cell. This method allows the selection of only a limited number of aptamers and only further to a high number of selection and/or counter-selection cycles. In addition, the selected aptamers display low cell specificity and are able to discriminate between cells of distant tumor types only (T-cell versus B cell lymphoma or small lung cancer cell versus large cell lung cancer, two cancer types of different origin).
Therefore, there is the need to provide a simplified method for obtaining aptamers comprising fewer cycles and resulting in aptamers with high specificity, even able to discriminate between different cells of the same tumor type, possessing different phenotypes (different resistance to a given physical or chemical therapeutic drug, different tumor mass growth properties, different ability to metastasize and different malignancy).
The authors of the present invention have already generated specific aptamers for the human receptor tyrosine kinase, Ret (Cerchia et al., 2005; WO 2005/093097), however they cannot be used to solve the problem of the invention.
The present invention discloses a simplified method to generate nucleic acid-based aptamers that bind to cancer cell-surface epitopes as unique tools to identify a surface molecular signature of cancer cells and thus permits to generate a small panel of high specific ligands capable of distinguish between even two closely related cell types. This approach, based on the use of living cells as target for the aptamers selection (whole-cell SELEX), allows selecting aptamers in a physiological context, and, most importantly, can be done without prior knowledge of the target molecules. The methods include much fewer steps than prior art methods. In addition and by contrast to the method of the application WO 2008/019142, the present protocol is specifically designed to target epitopes that are not internalised in the cell: ie short time of incubation of the library with cells are used and no trypsin treatment is performed. The present approach permits to identify and validate new tumor biomarkers.
The nucleic acid-based aptamers of the invention are able to discriminate between malignant and non malignant cell phenotype. The aptamers can also discriminate two different phenotypes within the same tumor cell type as for example, the resistance to a given physical or chemical therapeutic drug, the growth properties of the tumor mass, the ability to metastasize and the malignancy. The panel of aptamer molecules obtained and obtainable with the method of the present invention represent an innovative tool to detect cell surface specific epitopes as a signature of cancer cells in terms of tumor type, malignancy, therapeutic response, metastatic potential, proliferation and apoptotic rate. The panel of aptamer molecules obtained and obtainable with the method of the present invention represent an innovative tool to specifically target cancer cell with given surface specific epitopes in terms of tumor type, malignancy, therapeutic response, metastatic potential, proliferation and apoptotic rate.